Bi-allelic Mutations in PKD1L1 Are Associated with Laterality Defects in Humans.

Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans.

A genome-wide association study of congenital cardiovascular left-sided lesions shows association with a locus on chromosome 20.

Congenital heart defects involving left-sided lesions (LSLs) are relatively common birth defects with substantial morbidity and mortality. Previous studies have suggested a high heritability with a complex genetic architecture, such that only a few LSL loci have been identified. We performed a genome-wide case-control association study to address the role of common variants using a discovery cohort of 778 cases and 2756 controls. We identified a genome-wide significant association mapping to a 200 kb region on chromosome 20q11 [P= 1.72 × 10-8 for rs3746446; imputed Single Nucleotide Polymorphism (SNP) rs6088703 P= 3.01 × 10-9, odds ratio (OR)= 1.6 for both]. This result was supported by transmission disequilibrium analyses using a subset of 541 case families (lowest P in region= 4.51 × 10-5, OR= 1.5). Replication in a cohort of 367 LSL cases and 5159 controls showed nominal association (P= 0.03 for rs3746446) resulting in P= 9.49 × 10-9 for rs3746446 upon meta-analysis of the combined cohorts. In addition, a group of seven SNPs on chromosome 1q21.3 met threshold for suggestive association (lowest P= 9.35 × 10-7 for rs12045807). Both regions include genes involved in cardiac development-MYH7B/miR499A on chromosome 20 and CTSK, CTSS and ARNT on chromosome 1. Genome-wide heritability analysis using case-control genotyped SNPs suggested that the mean heritability of LSLs attributable to common variants is moderately high ([Formula: see text] range= 0.26-0.34) and consistent with previous assertions. These results provide evidence for the role of common variation in LSLs, proffer new genes as potential biological candidates, and give further insight to the complex genetic architecture of congenital heart disease.

Rare variants in NR2F2 cause congenital heart defects in humans.

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters.

Exome sequencing identifies rare variants in multiple genes in atrioventricular septal defect.

PURPOSE: The genetic etiology of atrioventricular septal defect (AVSD) is unknown in 40% cases. Conventional sequencing and arrays have identified the etiology in only a minority of nonsyndromic individuals with AVSD. METHODS: Whole-exome sequencing was performed in 81 unrelated probands with AVSD to identify potentially causal variants in a comprehensive set of 112 genes with strong biological relevance to AVSD. RESULTS: A significant enrichment of rare and rare damaging variants was identified in the gene set, compared with controls (odds ratio (OR): 1.52; 95% confidence interval (CI): 1.35-1.71; P = 4.8 × 10(-11)). The enrichment was specific to AVSD probands, compared with a cohort without AVSD with tetralogy of Fallot (OR: 2.25; 95% CI: 1.84-2.76; P = 2.2 × 10(-16)). Six genes (NIPBL, CHD7, CEP152, BMPR1a, ZFPM2, and MDM4) were enriched for rare variants in AVSD compared with controls, including three syndrome-associated genes (NIPBL, CHD7, and CEP152). The findings were confirmed in a replication cohort of 81 AVSD probands. CONCLUSION: Mutations in genes with strong biological relevance to AVSD, including syndrome-associated genes, can contribute to AVSD, even in those with isolated heart disease. The identification of a gene set associated with AVSD will facilitate targeted genetic screening in this cohort.

Recommendations for the integration of genomics into clinical practice.

The introduction of diagnostic clinical genome and exome sequencing (CGES) is changing the scope of practice for clinical geneticists. Many large institutions are making a significant investment in infrastructure and technology, allowing clinicians to access CGES, especially as health-care coverage begins to extend to clinically indicated genomic sequencing-based tests. Translating and realizing the comprehensive clinical benefits of genomic medicine remain a key challenge for the current and future care of patients. With the increasing application of CGES, it is necessary for geneticists and other health-care providers to understand its benefits and limitations in order to interpret the clinical relevance of genomic variants identified in the context of health and disease. New, collaborative working relationships with specialists across diverse disciplines (e.g., clinicians, laboratorians, bioinformaticians) will undoubtedly be key attributes of the future practice of clinical genetics and may serve as an example for other specialties in medicine. These new skills and relationships will also inform the development of the future model of clinical genetics training curricula. To address the evolving role of the clinical geneticist in the rapidly changing climate of genomic medicine, two Clinical Genetics Think Tank meetings were held that brought together physicians, laboratorians, scientists, genetic counselors, trainees, and patients with experience in clinical genetics, genetic diagnostics, and genetics education. This article provides recommendations that will guide the integration of genomics into clinical practice.Genet Med 18 11, 1075-1084.

The phenotypic spectrum of ZIC3 mutations includes isolated d-transposition of the great arteries and double outlet right ventricle.

Disease causing mutations for heterotaxy syndrome were first identified in the X-linked laterality gene, ZIC3. Mutations typically result in males with situs ambiguus and complex congenital heart disease; however affected females and one male with isolated d-transposition of the great arteries (d-TGA) have been reported. We hypothesized that a subset of patients with heart defects common to heterotaxy but without laterality defects would have ZIC3 mutations. We also sought to estimate the prevalence of ZIC3 mutations in sporadic heterotaxy. Patients with TGA (n = 169), double outlet right ventricle (DORV; n = 89), common atrioventricular canal (CAVC; n = 41), and heterotaxy (n = 54) underwent sequencing of ZIC3 exons. We tested 90 patients with tetralogy of Fallot (TOF) to correlate genotype with phenotype. Three potentially disease-related missense mutations were detected: c.49G > T (Gly17Cys) in a female with isolated DORV, c.98C > T (Ala33Val) in a male with isolated d-TGA, and c.841C > T (His281Tyr) in a female with sporadic heterotaxy. We also identified a novel insertion (CPFP333ins) in a family with heterotaxy. All were absent in 200 control patients and the 1000 Genomes Project (n = 629). No significant mutations were found in patients with TOF. Functional studies demonstrated reduced transcriptional activity of the ZIC3 His281Tyr mutant protein. ZIC3 mutations were rarely identified in isolated DORV and d-TGA suggesting that a subset of DORV and d-TGA may fall within the spectrum of laterality defects. ZIC3 mutations were found in 3.7% of patients with sporadic heterotaxy; therefore testing should be considered in patients with heterotaxy.

Genetic Diagnosis and the Severity of Cardiovascular Phenotype in Patients With Elastin Arteriopathy.

BACKGROUND: Elastin insufficiency causes recurrent vascular stenoses. Hemizygous deletion of the elastin gene (ELN) causes Williams-Beuren syndrome (WBS), while single nucleotide variants in ELN cause nonsyndromic supravalvar aortic stenosis (SVAS). Our objective was to compare cardiovascular disease outcomes in patients with WBS and nonsyndromic SVAS. METHODS: Patients (81 WBS, 42 nonsyndromic SVAS) with cardiovascular disease were included in this retrospective single center study. Freedom from surgical and catheter interventions and reinterventions was compared. Vascular tissue from 8 patients and 6 controls was analyzed for arterial wall architecture. RESULTS: Patients with nonsyndromic SVAS presented at a younger age (median 0.3 [0.4-0.7] years) compared with patients with WBS (1.3 [0.2-3.0] years) and had lower freedom from surgical/catheter interventions compared with patients with WBS, with median event-free survival 1.1 (0.3-5.9) versus 4.7 (2.4-13.3) years, respectively (hazard ratio, 1.62 [95% CI, 1.02-2.56]; P=0.04). Patients with nonsyndromic SVAS also had a lower freedom from reinterventions (P=0.054 by log-rank test). This was related in part to a higher frequency of primary and reinterventions for concomitant valvar aortic stenosis. Histology revealed abnormal intimal and medial thickening, disorganized and fragmented elastic fibers, reduced smooth muscle calponin expression, and increased macrophage marker, CD68, expression in the arterial walls in patients with WBS and nonsyndromic SVAS compared with controls. CONCLUSIONS: Patients with nonsyndromic SVAS require early and more frequent vascular and valvular interventions and reinterventions, in particular for concomitant valvar aortic stenosis compared with patients with WBS. This provides important prognostic information to guide counseling of affected families with cardiovascular disease and may guide primary intervention strategies based on predicted risk of restenosis.

Life-threatening flecainide intoxication in a young child secondary to medication error.

OBJECTIVE: To describe a case of life-threatening flecainide intoxication in a toddler, secondary to accidental reversal of syringes used for oral administration. CASE SUMMARY: A 2-year-old male with a history of a persistent junctional reciprocating tachycardia had been receiving flecainide 4.8 mg/kg/day (1 mL 3 times daily) and nadolol 2 mg/kg/day (5 mL once daily) for 10 months. One morning, 3 hours after the drugs were administered, he became bradycardic (heart rate 50 beats/min) and then presented to the emergency department with vital signs absent. After initial cardiopulmonary resuscitation and epinephrine, he was bradycardic; this was followed by wide-complex tachycardia that converted rapidly to narrow-complex tachycardia after bolus administration of intravenous sodium bicarbonate for suspected flecainide intoxication. Following resuscitation, he remained hemodynamically stable and was discharged in normal sinus rhythm without neurologic sequelae. Drug concentrations obtained at the time of presentation showed a serum concentration of flecainide of 0.668 microg/mL. Drug formulations were also analyzed and found to contain the expected concentration of flecainide. DISCUSSION: Literature regarding adverse drug events in the pediatric outpatient population is reviewed, as well as how these risks apply to flecainide, a medication with a low margin of safety. Pediatric experience with flecainide intoxication and sodium bicarbonate administration as an antidote is reviewed. Analysis of the serum drug concentrations demonstrated blood concentrations consistent with syringe reversal, which would have produced a 5-fold flecainide overdose. The Naranjo probability scale indicated a highly probable relationship between flecainide ingestion and the life-threatening event in this case. CONCLUSIONS: This case of life-threatening flecainide intoxication in a young child, secondary to accidental reversal of medication syringes, underscores the importance of providing parents with accurate dispensing information and labeling medication bottles and syringes in an unambiguous manner.

Genetic architecture of laterality defects revealed by whole exome sequencing.

Aberrant left-right patterning in the developing human embryo can lead to a broad spectrum of congenital malformations. The causes of most laterality defects are not known, with variants in established genes accounting for <20% of cases. We sought to characterize the genetic spectrum of these conditions by performing whole-exome sequencing of 323 unrelated laterality cases. We investigated the role of rare, predicted-damaging variation in 1726 putative laterality candidate genes derived from model organisms, pathway analyses, and human phenotypes. We also evaluated the contribution of homo/hemizygous exon deletions and gene-based burden of rare variation. A total of 28 candidate variants (26 rare predicted-damaging variants and 2 hemizygous deletions) were identified, including variants in genes known to cause heterotaxy and primary ciliary dyskinesia (ACVR2B, NODAL, ZIC3, DNAI1, DNAH5, HYDIN, MMP21), and genes without a human phenotype association, but with prior evidence for a role in embryonic laterality or cardiac development. Sanger validation of the latter variants in probands and their parents revealed no de novo variants, but apparent transmitted heterozygous (ROCK2, ISL1, SMAD2), and hemizygous (RAI2, RIPPLY1) variant patterns. Collectively, these variants account for 7.1% of our study subjects. We also observe evidence for an excess burden of rare, predicted loss-of-function variation in PXDNL and BMS1- two genes relevant to the broader laterality phenotype. These findings highlight potential new genes in the development of laterality defects, and suggest extensive locus heterogeneity and complex genetic models in this class of birth defects.

Whole exome sequencing in 342 congenital cardiac left sided lesion cases reveals extensive genetic heterogeneity and complex inheritance patterns.

BACKGROUND: Left-sided lesions (LSLs) account for an important fraction of severe congenital cardiovascular malformations (CVMs). The genetic contributions to LSLs are complex, and the mutations that cause these malformations span several diverse biological signaling pathways: TGFB, NOTCH, SHH, and more. Here, we use whole exome sequence data generated in 342 LSL cases to identify likely damaging variants in putative candidate CVM genes. METHODS: Using a series of bioinformatics filters, we focused on genes harboring population-rare, putative loss-of-function (LOF), and predicted damaging variants in 1760 CVM candidate genes constructed a priori from the literature and model organism databases. Gene variants that were not observed in a comparably sequenced control dataset of 5492 samples without severe CVM were then subjected to targeted validation in cases and parents. Whole exome sequencing data from 4593 individuals referred for clinical sequencing were used to bolster evidence for the role of candidate genes in CVMs and LSLs. RESULTS: Our analyses revealed 28 candidate variants in 27 genes, including 17 genes not previously associated with a human CVM disorder, and revealed diverse patterns of inheritance among LOF carriers, including 9 confirmed de novo variants in both novel and newly described human CVM candidate genes (ACVR1, JARID2, NR2F2, PLRG1, SMURF1) as well as established syndromic CVM genes (KMT2D, NF1, TBX20, ZEB2). We also identified two genes (DNAH5, OFD1) with evidence of recessive and hemizygous inheritance patterns, respectively. Within our clinical cohort, we also observed heterozygous LOF variants in JARID2 and SMAD1 in individuals with cardiac phenotypes, and collectively, carriers of LOF variants in our candidate genes had a four times higher odds of having CVM (odds ratio = 4.0, 95% confidence interval 2.5-6.5). CONCLUSIONS: Our analytical strategy highlights the utility of bioinformatic resources, including human disease records and model organism phenotyping, in novel gene discovery for rare human disease. The results underscore the extensive genetic heterogeneity underlying non-syndromic LSLs, and posit potential novel candidate genes and complex modes of inheritance in this important group of birth defects.

The prevalence of 16p12.1 microdeletion in patients with left-sided cardiac lesions.

SETTING: Left-sided cardiac lesions have a birth prevalence of approximately 1 in 1000 and have been shown to be heritable in pedigree studies. A large microdeletion at 16p12.1 is associated with childhood developmental delay, and initial studies describing this deletion identified left-sided lesions as an enriched phenotype compared with a control population. OBJECTIVE: The aim of this study is to determine whether patients with left-sided cardiac lesions have an increased frequency of 16p12.1 microdeletions as compared with control populations. DESIGN: A cohort of 262 probands with left-sided lesions, including 53 with isolated aortic stenosis/bicuspid aortic valve, 83 with coarctation of the aorta with or without aortic stenosis/bicuspid aortic valve, and 126 with hypoplastic left heart syndrome were assessed for copy number variation at 16p12.1. The control cohort included 595 patients with conotruncal defects as a cardiac control and 971 healthy children. RESULTS: We detected one patient in the left-sided lesion cohort with a large duplication partially overlapping the reported 16p12.1 microdeletion, along with one patient each in the conotruncal and control cohorts with a deletion in the same region. None of these patients had dysmorphic features, extracardiac malformations, or developmental delay. CONCLUSION: In our cohort, structural variation at 16p12.1 was not identified with increased frequency in patients with left-sided lesions as compared with controls.

High throughput exome coverage of clinically relevant cardiac genes.

BACKGROUND: Given the growing use of whole-exome sequencing (WES) for clinical diagnostics of complex human disorders, we evaluated coverage of clinically relevant cardiac genes on WES and factors influencing uniformity and depth of coverage of exonic regions. METHODS: Two hundred and thirteen human DNA samples were exome sequenced via Illumina HiSeq using different versions of the Agilent SureSelect capture kit. 50 cardiac genes were further analyzed including 31 genes from the American College of Medical Genetics (ACMG) list for reporting of incidental findings and 19 genes associated with congenital heart disease for which clinical testing is available. Gene coordinates were obtained from two databases, CCDS and Known Gene and compared. Read depth for each region was extracted from the exomes and used to assess capture variability between kits for individual genes, and for overall coverage. GC content, gene size, and inter-sample variability were also tested as potential contributors to variability in gene coverage. RESULTS: All versions of capture kits (designed based on Consensus coding sequence) included only 55% of known genomic regions for the cardiac genes. Although newer versions of each Agilent kit showed improvement in capture of CCDS regions to 99%, only 64% of Known Gene regions were captured even with newer capture kits. There was considerable variability in coverage of the cardiac genes. 10 of the 50 genes including 6 on the ACMG list had less than the optimal coverage of 30X. Within each gene, only 32 of the 50 genes had the majority of their bases covered at an interquartile range ≥30X. Heterogeneity in gene coverage was modestly associated with gene size and significantly associated with GC content. CONCLUSIONS: Despite improvement in overall coverage across the exome with newer capture kit versions and higher sequencing depths, only 50% of known genomic regions of clinical cardiac genes are targeted and individual gene coverage is non-uniform. This may contribute to a bias with greater attribution of disease causation to mutations in well-represented and well-covered genes. Improvements in WES technology are needed before widespread clinical application.

Situs inversus totalis and a novel ZIC3 mutation in a family with X-linked heterotaxy.

Disorders of laterality consist of a complex set of malformations resulting from failure to establish normal asymmetry along the left-right axis, and include both heterotaxy and situs inversus totalis. Zinc fingers in cerebellum 3 (ZIC3) was the first gene to be definitively associated with heterotaxy syndromes in humans (OMIM #306955), with 13 mutations previously described in both familial and sporadic cases. We now report the clinical and molecular characterization of a five-generation family originally reported in 1974 as having X-linked dextrocardia. Longitudinal follow-up revealed that this family has X-linked heterotaxy due to a missense mutation, c.1048A>G(R350G), in the third zinc finger domain of ZIC3. The pedigree demonstrates the first reported case of situs inversus totalis associated with a ZIC3 mutation in a male and the second reported case of incomplete penetrance in an unaffected transmitting male, as well as a wide range of phenotypes of varying severity. Several affected members also exhibit renal and hindgut malformations, consistent with previously reported secondary features in ZIC3 mutations. The spectrum of features in this family emphasizes the importance of thorough molecular and imaging studies in both sporadic and familial cases of heterotaxy to ensure accurate prenatal diagnosis and recurrence risk counseling.

Genetic testing in congenital heart disease: ethical considerations.

On March 16, 2012, the Ethics of the Heart 2012: Ethical and Policy Challenges in Pediatric and Adult Congenital Heart Disease Conference took place in Philadelphia, Pennsylvania. The first session focused on the ethics surrounding genetic testing in patients with congenital heart disease. Summarized here is the introductory presentation given by Dr Elizabeth Goldmuntz entitled "The Role of Genetic Testing in Congenital Heart Disease," followed by a case presentation given by Dr Lisa D'Alessandro. The case and the panel discussion that ensued highlight several ethical principles and challenges in this unique patient population.

Personalized medicine in the care of the child with congenital heart disease: discovery to application.

On October 27-28, 2012, the SickKids Labatt Family Heart Centre and the Heart Centre Biobank Registry hosted the second international GenomeHeart symposium in Toronto, Ontario. The symposium featured experts in cardiology, developmental biology, pharmacology, genomics, bioinformatics, stem cell biology, biobanking, and ethics. The theme of this year's symposium was the application of emerging technologies in genomics, proteomics, transcriptomics, and bioinformatics to diagnostics and therapeutics of the child with heart disease. Social, ethical, and economic issues were also discussed in the context of clinical translation. We highlight some of the themes that emerged from this exciting 2-day event.